Field of the Invention
The invention relates to the determination of microbial resistance to antibiotics, in particular those microbes which produce beta-lactamases.
Description of the Related Art
The publication WO 2011154517 A1 (M. Kostrzewa, K. Michelmann, K. Sparbier) and the equivalent publications DE 102010023452 B4 and US 20130095511 A1 present a method to determine microbial beta-lactamase resistances which is based on the mass spectrometric measurement of the enzymatic modification of beta-lactam antibiotics, or substrates with a similar structure, by microbial beta-lactamases. To this end, bacteria are introduced into solutions with beta-lactam antibiotics, or substrates with a similar structure, and incubated for around three hours, for example. The bacteria are then preferably separated out by centrifugation or filtration. One to two microliters of the solution containing no bacteria is subsequently applied to a mass spectrometric sample support, dried, and coated with a matrix solution for ionization by matrix-assisted laser desorption (MALDI). Measurement in a time-of-flight mass spectrometer with a MALDI ion source produces a mass spectrum from which it is possible to determine whether an enzymatically modified antibiotic or substrate is present, especially a hydrolytic splitting of the beta-lactam ring, which indicates that the microbes are resistant.
A similar method is presented in the publication WO 2012/023845 A1 (Luider et al.), where in general the modifications of antibiotics by microbial enzymes are determined by mass spectrometry.
The methods described in these documents have a relatively large number of individual steps, especially centrifugation or filtration, which takes a lot of time and often involves manual procedures. The known methods normally take around three hours. There continues to be a need for methods to determine microbial resistances which involve less work, are easier to automate and faster, and allow a high sample throughput with smaller sample quantities.
There is an ongoing need to provide methods and instruments for determining a microbial resistance, which is based on an enzymatic modification of antibiotics, by mass spectrometry wherein the methods comprise only a few individual steps and preferably last less than an hour.